P550Mutations in thin filament proteins that cause familial dilated cardiomyopathy uncouple troponin I phosphorylation from changes in myofibrillar Ca2+ sensitivity

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The pure form of familial dilated cardiomyopathy (DCM) is mainly caused by mutations in sarcomeric proteins. Studies of cardiac muscle thin filaments with DCM mutations using recombinant mutant troponin I (Tn I), troponin T (Tn T) or troponin C (Tn C) and α-tropomyosin suggested that DCM mutations caused a 2-3 fold lower Ca2+ sensitivity, but measurements using natively phosphorylated troponin failed to show any consistent changes in Ca2+ sensitivity due to DCM mutations (a review of publications showed that 6 out of 14 DCM mutations studied did not have lower Ca2+ sensitivity). In contrast to this we consistently observed that DCM mutations affect the relationship between Ca2+ sensitivity and Tn I phosphorylation by PKA. Normally, PKA phosphorylation of Tn I causes a 2-3 fold decrease in myofibrillar Ca2+ sensitivity that is important for the lusitropic response to β-adrenergic stimulation. Using quantitative in vitro motility assays and manipulating the phosphorylation level with PKA or phosphatase, we found that in thin filaments containing DCM mutations Ca2+ sensitivity was usually low and did not change with the level of Tn I phosphorylation. We have now demonstrated this uncoupling effect with mutations in every component of the thin filaments (E361G in cardiac actin, E40K and E54K in α-tropomyosin, G159D in cTn C, R141W and ΔK210 in cTn T and K36Q in cTn I). Uncoupling was found with native mutant protein from human and mouse heart and with recombinant mutant protein expressed with baculovirus/sf9 systems. Uncoupling was found to be independent of the fraction of mutated protein present in the range 45 to 100%. We propose that DCM mutations in thin filament proteins uncouple myofilament Ca2+ sensitivity from troponin phosphorylation and blunt the response to β-adrenergic stimulation, leading to reduced cardiac reserve with consequent contractile dysfunction under stress.

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